Current Health Sciences Journal Vol. 44, No. 3, 2018 July-September Original Paper Histopathological Aspects of the Myocardium in Dilated Cardiomyopathy RADU MITRUT1, ALEX EMILIAN STEPAN2, DANIEL PIRICI3

1PhD student, Department of , University of Medicine and Pharmacy of Craiova 2Department of Pathology, University of Medicine and Pharmacy of Craiova 3Department of Research Methodology, University of Medicine and Pharmacy of Craiova

ABSTRACT: Dilated cardiomyopathy is the most common form of , accounting for approximately 60% of all cardiomyopathies. We proposed to identify histopathological changes of the myocardium in dilative cardiomyopathy. This study comprised a total of 19 cases, represented by myocardial fragments from deceased patients with diagnosis of dilated cardiomyopathy. Histopathological analysis allowed changes to be observed for both myocytes and myocardial interstitial components. We have found a combination of hypertrophic, atrophic and normal myocardocytes, or associated with the presence of hydropic changes. We rarely identified the aspect of myocytosis, cytoplasmic accumulation of pigment or mucinous material, and variable nuclear pleomorphism. At the interstitial level we noticed changes in fibrosis, lipomatosis and rarely the presence of inflammatory infiltrate. Histopathological characteristics of the myocardium in dilated cardiomyopathy are numerous but nonspecific, similar to those in the terminal stages of other cardiac . KEYWORDS: Dilated myocardiopathy,

Introduction Material and Methods Cardiomyopathies are defined as diseases of This study included a total of 19 cases from the myocardium with associated structural and patients diagnosed with dilated cardiomyopathy functional abnormalities, which are classified diagnosed in the Cardiology Clinic of traditionally according to morphological and Emergency County Hospital Craiova. functional criteria into four categories: dilated The studied material was represented by cardiomyopathy, hypertrophic cardiomyopathy, myocardial fragments harvested during necropsy, restrictive cardiomyopathy and arrhythmogenic which were fixed in 10% formalin and then right ventricular cardiomyopathy/. processed by the classic histopathological Dilated cardiomyopathy is the most common technique using the BioOptica tissue processor and form of heart muscle disease, comprising hematoxylin-eosin (HE), periodic acid-Schiff approximately 60% of all cardiomyopathies (PAS)-Alcian Blue (AB) and Masson's trichrome [1,2]. staining. It is a progressive, diffuse process involving We have followed a number of cardiomyocytes from both ventricles, which histopathological parameters such as myocyte eventually become dilated and dysfunctional. , vacuolar degeneration, myocytolysis, The myocardium relaxes, which means it lipofuscin accumulation, mucin accumulation, stretches and becomes thinner, and consequently interstitial fibrosis, lipomatosis and the ventricular chamber widens. It is a inflammatory infiltrate. progressive, diffuse process involving Each of these parameters was rated as focal cardiomyocytes from both ventricles, which when it was ≤10%, zonal 10-25% and extended eventually become dilated and dysfunctional. The ≥25%. Interpretation was done using the Nikon myocardium relaxes, which means it stretches and microscope Eclipse E600 and software program becomes thinner, and consequently the ventricular Lucia 5. The written informed consent was chamber widens. The histologic features of dilated obtained from relatives or caretakers of all cardiomyopathy are nonspecific; therefore, it is a patients included in this study. microscopic diagnosis of exclusion [3] and identified in various studies only in myocardium of Results patients with end-stage disease [4]. Histopathological features of myocardium The present study aims to identify the spread from the 19 patients with dilated of histopathological changes of myocardiocytes cardiomyopathy diagnosis allowed changes to and myocardial interstitium in patients with be observed for both myocytes and myocardial dilated cardiomyopathy. interstitial components (Table 1).

10.12865/CHSJ.44.03.07 243 Radu Mitrut et al. - Histopathological Aspects of the Myocardium in Dilated Cardiomyopathy

Table 1. Distribution of cases according to histopathological parameters

Histopathological Focal Zonal Extended parameters Number % Number % Number % of cases of cases of cases Myocytes atrophy 3 15.8 11 57.8 5 26.4 Vacuolar degeneration 10 52.6 7 36.8 2 10.5 Myocytolysis 7 36.8 0 0 0 0 Lipofuscin accumulation 9 47.3 0 0 0 0 Mucin accumulation 6 31.5 1 5.2 0 Nuclear pleomorphism 5 26.4 3 15.8 1 5.2 Interstitial fibrosis 11 57.8% 6 31.5% 2 10.5% Lipomatosis 8 42.1% 5 26.3% 0 0 Inflammatory infiltrate 5 26.4% 0 0 0 0

Myocytes had a normal architectural variable spread. In 10 cases (53%) the distribution, but showed changes in their appearance was only focal, affecting a small number, size, and shape, as well as changes in number of myocytes. In 7 cases (37%) the the cytoplasm and nucleus. changes included larger areas with a zonal As for the size of myocytes, there were quite character, and in 2 cases it was diffused into the large variations. We have observed a myocardial fibers (10%). Changes in vacuolar combination of hypertrophic, atrophic and degeneration had variable cytoplasmic normal myocardiocytes, or associated with the morphology and topography. presence of degenerative changes. Most often, we observe the presence of We have identified atrophy in all investigated several empty vacuoles, with irregular contours cases, but with varying extent. and imprecisely delimited, isolated or confluent, The most common atrophy was present with dispersed in the cytoplasm (Fig.2). zonal character (11 cases, 57.8%), rarely was extensive or focal aspect. Due to the atrophy of the myocytes they had the appearance of long and thin fibers, with a sinuous path, with a characteristic appearance of "corrugated fibers" (Fig.1), separated from each other by loose spaces, a suggestive aspect for edematous infiltration of the interstitia.

Fig.2. Dilated cardiomyopathy, hydropic degeneration, HE staining, x400

Myocytolysis was rarely identified, recognized by muscle fibers fragmentation. The lesion had only focal character, affecting isolated myocytes or small groups. In 9 cases we noted the accumulation of the lipofuscin pigment in the form of brown Fig.1. Dilated cardiomyopathy, atrophy with "corrugated" fibers appearance, HE staining, x10 granules of variable size, preferentially perinuclear disposed. The cytoplasmic Myocytes cytoplasm has shown some accumulation of mucinous material, with changes, such as vacuolar degeneration, slightly basophilic tinctoriality, PAS positive, myocytosis, reduction in myofibril counts, was rarely present (7 cases, 37%) and almost lipofuscin or mucin pigment accumulation. always focal, only in one case the modification Changes in vacuolar degeneration were being zonal (Fig.3, Fig.4). present in all investigated cases but with

244 10.12865/CHSJ.44.03.07 Current Health Sciences Journal Vol. 44, No. 3, 2018 July-September Fibrosis was diffuse or focal, with interstitial or sometimes perivascular topography. In interstitial fibrosis, we noticed the presence of the fibrillar collagen in intermuscular spaces that are normally devoid of collagen. The distribution pattern was quite variable, from a fine peri-myocyte distribution to massive scars, sometimes similar to those in ischemic cardiopathy. Collagen has individually surrounded the myocardial fibers that have a "ragged" appearance due to the corrugated cell membrane. In the case of perivascular fibrosis, collagen has accumulated in the adventitia of intramedullary coronary arteries and arterioles, Fig.3. Dilated cardiomyopathy, mucinous otherwise untouched. At the interstitial level we degeneration, HE staining, x400 noticed changes in fibrosis, lipomatosis and rarely the presence of inflammatory infiltrate. Depending on the magnitude of the change, the analyzed cases presented minimal fibrosis with a focal character in 11 cases, moderate that had a zonal character in 6 cases, or extended and diffuse in 2 cases. The aspect was subsequently confirmed by Masson's trichrome staining (Fig.6, Fig.7).

Fig.4. Dilated cardiomyopathy, mucinous degeneration, PAS staining, x400

Myocytes nuclei also had changes, both in form and in tinctoriality. Their dimensions were variable, sometimes large and hyperchromatic, with bizarre forms and variable pleomorphism from mild (5 cases) to moderate (3 cases) and to Fig.6. Dilated cardiomyopathy, moderate fibrosis severe (1 case) (Fig.5). and diffuse hydropic degeneration, HE staining, x100

Fig.5. Dilated cardiomyopathy, nuclear pleiomorphism and perinuclear lipofuscin, Fig.7. Dilated cardiomyopathy, moderate fibrosis, HE staining, x400 Masson's trichrome staining, x40

At the interstitial level we noticed changes in Sometimes, we noticed the presence of fatty fibrosis, lipomatosis and rarely the presence of tissue infiltrate (lipomatosis) with focal inflammatory infiltrate. character. The appearance was identified in

10.12865/CHSJ.44.03.07 245 Radu Mitrut et al. - Histopathological Aspects of the Myocardium in Dilated Cardiomyopathy 13 cases, where adipocytes, generally of small from dilated cardiomyopathy being explained by size, were disposed isolated or in small groups the progressive degeneration of cardiomyocytes between the myocardial fibers. [9]. Inflammatory infiltrate composed of rare Myocytic atrophy is frequently mentioned lymphocytes was rarely identified, with a between the histopathological features of dilated dispersed appearance in fibro-collagen tissue. cardiomyopathy [6], being defined by the We also identified rare mast cells, confirmed by presence of elongated, thin myocytes with a the PAS-AB staining (Fig.8). narrow cytoplasmic border, their corrugated appearance constituting an indicative image of their overextension. Corrugated myocytes, especially when associated with focal edema, are characteristic changes in acute myocardial [10], but have also been described in patient with dilated cardiomyopathy [11,12]. In our study, we observed quite large variations in the myocardial size, along with the atrophic myocyte areas characterized by the reduction of the cross-sectional diameter, as well as the areas with normal or sometimes even hypertrophic myocytes. In one study, the percentage of myocytes decreased from 78.10±7.34 in control Fig.8. Dilated cardiomyopathy, rare mastocytes, Alcian Blue PAS staining, x400 cases to 63.39+/-9.22 in dilated cardiomyopathy (p<0.05) and 47.28+/-15.01 in arrhythmogenic Discussions right ventricular cardiomyopathy (p<0.01) [13]. We identified atrophy of myocardial cells in The main components of myocardial all investigated cases, but with varying structure are cardiomyocytes and the distribution, respectively focal length in 15.8% extracellular matrix (EM). Cardiomyocytes of cases, with zonal character in 57.8% of cases account for approximately 76% of myocardial and diffuse appearance in 26.4% of these. volume and the extracellular matrix is Yonesaka et al. reported myocardial atrophy in approximately 24% [5]. In dilated only 2 out of 16 patients with idiopathic dilated cardiomyopathy, both components of both cardiomyopathy studied [14]. Another study myocardial and extracellular matrix were reported in dialyzed patients with dilated identified. The aspects described on cardiomyopathy: severe of endomyocardial biopsies from patients with myocytes with a mean myocytic diameter of dilated cardiomyopathy range from minimal 37.6±10.5 vs. 25.6+/-7.7 in control cases differences in the size of myocytes to those (p=0.001) and disruption of myocytes in 30% of typical of the disease with marked variations in cases of dilated cardiomyopathy [15]. the size of myofibrils, loss of myofibrils and Normal myocardiocytes have a length of 100 interstitial fibrosis [3,6]. In one study, the µm and a diameter of 10-25µm [16,17]. authors reported the association of interstitial Myocardial lesions associated with corrugated fibrosis with hypertrophy of myofibrils, along atrophy of myocytes consist of the presence of with degeneration and atrophy of the myocytes myocardial cells with a diameter of less than in 60% of the investigated dilated 10µm, with a corrugated appearance, which cardiomyopathy [7]. comprises at least half the thickness of the The content of fibrous tissue in the myocardial specimens in the upper and lower myocardium of patients with dilated portions of the left ventricle (LV). In one study, cardiomyopathy is associated with a the authors reported an average diameter of combination of hypertrophic, atrophic and 22.89µm (3.30) for myocardocytosis in dilated normal myocardiocytes or with the presence of cardiomyopathy patients compared to 16.74µm degenerative changes, which led to the (1.17) in control cases [18]. hypothesis of a vicious circle [8]. The vacuolar degeneration of myocytes is According to it, modified myocytes can frequently mentioned, with the appearance of release substances that stimulate the cytoplasmic vacuoles or sometimes as a extracellular matrix growth but in turn its perinuclear halo [19]. extension may overload myocytes and the continuous increase of the collagen network

246 10.12865/CHSJ.44.03.07 Current Health Sciences Journal Vol. 44, No. 3, 2018 July-September In the study, the presence of vacuolar matrix affects the mechanical properties of the degeneration of myocytes was observed in all myocardium during systole and diastole, and cases, often focal. Myocitolysis is not a alterations that lead to myocardial fibrosis, even characteristic change in dilated cardiomyopathy in intact cardiomyocytes, may lead to heart [3]. failure [24], perhaps due to limited supply of Johnson et al. did not find any case of dilated oxygen to cardiomyocytes and distortion of cardiomyopathy with myocytosis in cases of electrical contacts between them. endomyocardial biopsy, but the appearance was Quantification of collagen showed up to identified and reported in 9% of necropsy cases 4-fold increase from normal collagen volume, [20]. with a decrease in mature reticulated collagen, In the study we observed the presence of correlated with increased neutrophil collagenase myocytolysis in only 7 cases and it was only activity [25]. focal. After other studies in advanced stages of the Nuclear pleomorphism is frequently disease, the collagen concentration is increased described, with myocytes having dysmetric and by two [26] to five times [27]. dysmorphic nuclei [6,19]. Another study reported a percentage of For the analyzed cases we noticed the nuclear 18.71% (7.76) fibrosis in patients with dilated pleomorphism in 9 of the analyzed cases, cardiomyopathy compared to 13.39% (9.55) in frequently with focal character. Arbustini et al. normal patients [18]. analyzed the large nuclei and bizarre The percentage of collagen volume in the LV morphology, and found that the width of the myocardium is significantly higher in patients myocytes, nuclear diameter, and with dilated cardiomyopathy compared to those nucleo-sarcoplasmic ratio were significantly with ischemic cardiomyopathy [1]. higher in the group compared to cases lacking The increase in the collagen fraction is these modifications [21]. correlated with the degree of LV dilatation [28]. The mean values were 36+/-5µm, 14+/-3µm One study reported a correlation between the and 0.41+/-0.08 respectively for the cases with percentage of fibrosis, the LV ejection fraction nuclear changes, compared to 20+/-8µm, and the functional heart rate [29]. 7+/-2µm and 0.37+/-0.08 for cases without In cases where fibrosis was <10%, there were nuclear modification [21]. high ejection fraction values and are included in In one study, the authors reported statistically class I of global functional capacity, while cases significant differences in the number of with fibrosis >10% are in Class III and IV of nuclei/mm2 of myocardial tissue, diameter and global functionality capacity and have low area [18]. ejection fraction [29]. In another study it was suggested that nuclear Johnson et al. reported that fibrosis in abnormalities may constitute the main event in patients with dilated cardiomyopathy was in the of dilated cardiomyopathy, only 28% of endomyocardial biopsy specimens with authors reporting lowered nuclear density and in 50% of necropsy sections [20]. (18%, p<0.05), but the area of the nuclear In contrast, Sekiguchi et al. found the profile significantly increased (85%, p<0.001), presence of fibrosis in 100% of cases in both the nucleus/cytoplasmic relationship being thus endomyocardial biopsy and necropsy specimens modified [22]. [30]. Interstitial changes have been described in The volume of interstitial tissue density was several studies in patients with dilated nearly twice as high in patients with dilated cardiomyopathy. These consist of fibrosis, focal cardiomyopathy who died 3 years after surgery fat infiltration, and sometimes the presence of (reductive annuloplasty) compared to patients focal inflammatory infiltration. who survived 7 years after this time (0.20±0.023 The most typical change in dilated times 0.13±0.02, p<0.01) [31]. cardiomyopathy is the development of Another study reported extensive fibrosis in interstitial and perivascular fibrosis of varying dialysis patients with dilated cardiomyopathy, degrees [6,23]. with a mean area of LV fibrosis of 22.3+/-18.4% Fibrosis in dilated cardiomyopathy, as in vs. 21.3+/-14.6% [15]. other cardiac diseases, is an essential component The rate of non-fatal survival at 3 years for of the myocardial remodeling process but also dialysis patients with severe fibrosis (over 30%) has some distinctive features. Obviously, the was 42%, while for patients without severe composition and properties of the extracellular fibrosis it was 82% (p=0.03), the authors

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Corresponding Author: Alex Emilian Stepan, Department of Pathology, University of Medicine and Pharmacy of Craiova, 66 1 May Avenue, 200628 Craiova, Romania, e-mail: [email protected]

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